Interferon gamma (IFN-gamma) plays a crucial role in regulation of the immune system and is active on virtually all tissues. Its actions are mediated by its receptor that is the subject of this application. We previously identified two chains (IFN-gammaR1 and IFN-gammaR2) that are required for receptor activity. The current proposal focuses on the structure of the chains of the IFN-gamma receptor complex in live cells in the presence and absence of ligand. The specific aims directed to these goals are: to determine if all the IFN-gamma, receptor chains preassociated; the role of the Jak kinases in preassembly; determination of the sites on the receptor chains for preassembly; role of Stat1 in movement of the intracellular domains; determination of the kinetics of change in receptor structure after ligand binding; analysis of the kinetics and interactions of the downstream signaling components with the receptor complex; determine the sites of the receptor that serve as sites for cross-talk between the IFN-gamma and IFN-alpha receptor complexes. The configuration of the receptor chains will be studied by fluorescence resonance energy transfer in live cells. Because IFN-gamma plays a critical role in cellular and immune function, understanding all these aspects of its action will lead to better strategies to attack a variety of diseases such as cancers, and infectious and autoimmune diseases. Further application of this technology should lead to new rapid methods for high throughput screening and for identifying protein-protein interactions in live cells in real-time.